Diabetes Volume 69, November 2020 2503 Epigenetic Changes in Islets of Langerhans Preceding the Onset of Diabetes Meriem Ouni,1,2 Sophie Saussenthaler,1,2 Fabian Eichelmann,2,3 Markus Jähnert,1,2 Mandy Stadion,1,2 Clemens Wittenbecher,2,3,4 Tina Rönn,5 Lisa Zellner,1,2 Pascal Gottmann,1,2 Charlotte Ling,5 Matthias B. Schulze,2,3,6 and Annette Schürmann1,2,6 Diabetes 2020;69:2503–2517 | https://doi.org/10.2337/db20-0204 GENETICS/GENOMES/PROTEOMICS/METABOLOMICS The identification of individuals with a high risk of de- Alarming incident rates worldwide are projected to in- veloping type 2 diabetes (T2D) is fundamental for pre- crease the current prevalence of type 2 diabetes (T2D) vention. Here, we used a translational approach and from 422 million to 592 million in 2035 (1). T2D is prediction criteria to identify changes in DNA methyla- a progressive, chronic disorder with a long asymptomatic tion visible before the development of T2D. Islets of phase, averting detection for many years (2,3). Better Langerhans were isolated from genetically identical disease management might be possible with earlier de- 10-week-old female New Zealand Obese mice, which tection through robust, sensitive, and easily accessible differ in their degree of hyperglycemia and in liver fat content. The application of a semiexplorative ap- biomarkers of T2D. proach identified 497 differentially expressed and T2D is characterized by chronic hyperglycemia, which is methylated genes (P 5 6.42e-09, hypergeometric test) caused by an impaired insulin secretion from pancreatic enriched in pathways linked to insulin secretion and b-cells and an insulin resistance of target tissues. Aging, extracellular matrix-receptor interaction. The compar- a sedentary lifestyle, and obesity contribute to insulin re- ison of mouse data with DNA methylation levels of sistance. After long-term exposure to elevated lipid and incident T2D cases from the prospective European Pro- glucose levels, pancreatic islet function decreases (4,5), which spective Investigation of Cancer (EPIC)-Potsdam co- leads to insufficient compensation and a loss of b-cells. hort, revealed 105 genes with altered DNA methylation The involvement of epigenetic mechanisms in T2D at 605 cytosine-phosphate-guanine (CpG) sites, which development emerged as a promising research area (6). were associated with future T2D. AKAP13, TENM2, One epigenetic modification is DNA methylation, which CTDSPL, PTPRN2,andPTPRS showed the strongest mainly occurs at the 59 carbon of cytosine-phosphate- predictive potential (area under the receiver operating guanine (CpG) sites (7,8). DNA methylation marks are characteristic curve values 0.62–0.73). Among the new established during prenatal and early postnatal develop- candidates identified in blood cells, 655 CpG sites, ment and function throughout life to maintain the diverse located in 99 genes, were differentially methylated in gene expression patterns of different cell types (9,10) but islets of humans with T2D. Using correction for multiple can also arise later in somatic cells either by random events testing detected 236 genes with an altered DNA meth- or under the influence of the environment (11,12). Thus, ylation in blood cells and 201 genes in diabetic islets. tissue specificity and flexibility of DNA methylation in Thus, the introduced translational approach identified response to the environment are two major problems to novel putative biomarkers for early pancreatic islet face in order to identify stable epigenetic biomarkers of aberrations preceding T2D. disease risk. The aim of our translational study was to 1Department of Experimental Diabetology, German Institute of Human Nutrition Received 9 March 2020 and accepted 9 August 2020 Potsdam-Rehbruecke, Brandenburg, Germany This article contains supplementary material online at https://doi.org/10.2337/ 2 German Center for Diabetes Research, München-Neuherberg, Germany figshare.12794957. 3Department of Molecular Epidemiology, German Institute of Human Nutrition S.S. and F.E. equally contributed to the work. Potsdam-Rehbruecke, Brandenburg, Germany 4Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA © 2020 by the American Diabetes Association. Readers may use this article as 5Department of Clinical Sciences, Lund University, Malmö, Sweden long as the work is properly cited, the use is educational and not for profit, and the 6Institute of Nutritional Science, University of Potsdam, Potsdam, Germany work is not altered. More information is available at https://www.diabetesjournals .org/content/license. Corresponding author: Annette Schürmann, [email protected] 2504 Epigenetic Marks and Type 2 Diabetes Risk Diabetes Volume 69, November 2020 identify early epigenetic marks related to T2D by uncov- protocol, with additional DNase treatment. RNA samples ering methylome alterations in pancreatic islets of mice with RNA integrity number $8 (Agilent Bioanalyzer) were that occur before the onset of severe hyperglycemia and selected for RNA-seq. Transcriptome sequencing was carried assessing prospective T2D risk information conveyed by out by GATC biotech (Konstanz, Germany) on an Illumina congruent differential methylation in human blood. HiSeq platform. Adapters were trimmed and reads filtered for quality by using the wrapper Trim Galore! v0.4.2 and RESEARCH DESIGN AND METHODS Cutadapt 1.9.1 with option phred33. FastQC v0.11.5 was Animals, Diets, and Experimental Design used to check sample quality. Alignment of reads to reference A full description of animals and diets was detailed pre- genome was performed with HISAT2 v2.1.0, and fragments viously (13,14). At 5 weeks of age, female New Zealand per kilobase of exon model per million reads mapped values Obese (NZO) mice were placed on a high-fat diet (HFD) for transcripts was determined by Cufflinks 2.2.1, both with (20% kcal protein, 20% kcal carbohydrate, 60% kcal fat; default options for paired reads. We considered only tran- D12492; Research Diets) for 5 weeks. Five weeks after scripts with fragments per kilobase of exon model per million switching the diet, mice were killed during midlight cycle reads mapped mean values .1/group. Kyoto Encyclopedia of with acute exposure to isoflurane (Fig. 2A). Animal studies Genes and Genomes (KEGG) analysis was performed by the were approved by the animal welfare committees of the using DAVID 7 tool (16), with cutoff enrichment score set German Institute of Human Nutrition Potsdam-Rehbruecke to .1.7 and enriched P , 0.05. Network analysis was and local authorities (Landesamt für Umwelt, Gesundheit obtained with Ingenuity Pathway Analysis (IPA) (QIAGEN) und Verbraucherschutz, Brandenburg, Germany). (Supplementary Table 1). Pancreatic Islet Isolation WGBS in Pancreatic Islets Islet isolation was performed as described (15). Islets isolated Genomic DNA from NZO islets was isolated using Invisorb fromtwotofourmice(30–110 islets/mouse) were pooled Genomic DNA Kit II. One microgram of genomic DNA per sample for RNA sequencing (RNA-seq) and whole- from each pool was bisulfite converted (Zymo Research genome bisulfite sequencing (WGBS). The total number Corporation, Irvine, CA), and library preparation and of islets used for nucleic acid extraction was 900 and 1,500 sequencing steps were carried out by GATC. WGBS data for RNA and DNA, respectively. For RNA-seq of diabetes- in fastq format were generated using an Illumina HiSeq resistant (DR) mice, four individual islet pools were used platform for further analyses. Raw data have been quality that contained islets from 2 mice/pool; for diabetes-prone controlled and processed using Trim Galore! v0.4.2, (DP)mice, five pools were used that contained 2–3 mice/ FastQC v0.11.5, Bismark v0.17.08 (17), and MethPipe pool. WGBS was performed with five pools per group v3.4.2 (18) (Supplementary Table 1). from 4 animals/pool (Supplementary Table 1). Thus, A reference genome file was generated by combining each sample of islet pools comprised islets from differ- a GRCm38.68 B6 reference and a GRCm38p4 single nu- ent mice. cleotide polymorphism file in order to exchange all B6 with NZO high-quality single nucleotide polymorphisms. Meth- Blood Glucose, Body Weight, Body Composition, and ylation counting was carried out with MethPipe v3.4.2 Liver Fat Content default options. Body weight and blood glucose were measured from 7:00 Nonsymmetric CpG sites have been withdrawn, P values to 9:00 A.M. on a weekly basis by using a Contour blood have been calculated using log-likelihood ratio test. For the glucose meter (Bayer). At 5, 7, and 10 weeks of age, body final analysis, CpG sites that fit to the following criteria composition and liver fat content were analyzed using were used: 1) at least four of five samples with read counts nuclear magnetic resonance and computed tomography as in both groups, 2) average of read counts per group .20, described (14). and 3) SD per group .0 in both groups. Plasma Analysis Plasma adiponectin and leptin levels were measured by DNA Methylation Analysis in Human Blood Cells Mouse Adiponectin/Acrp30 (DY1119; R&D Systems) and The study sample is a nested case-control study derived from Mouse/Rat Leptin (MOB00; R&D Systems) ELISA kits, the prospective European Prospective Investigation into 5 respectively. Plasma triglycerides (T2449, F6428, G7793; Cancer and Nutrition (EPIC)-Potsdam cohort study (n Sigma), free fatty acids (91096, 91898, 91696; Wako), 27,548) designed to estimate the association of baseline cholesterol (10017, HUMAN), ALT (12212, HUMAN), AST measurements to